Integrins are cell adhesion receptors that transmit bidirectional signals across the plasma membrane and link the extracellular environment of a cell to the actin cytoskeleton. The conformation of the integrin extracellular domain and its affinity for ligand are dynamically regulated by a process termed “inside-out signaling.” Rapid upregulation of adhesiveness of integrins on platelets and white blood cells mediates hemostasis and leukocyte trafficking to sites of inflammation. By coupling to the actin cytoskeleton, integrins promote firm adhesion and provide traction for lamellipodium protrusion and locomotion. In migrating cells the adhesiveness of integrins is spatially and temporally regulated so that integrins are activated near the leading edge to support lamellipod extension and deactivated near the trailing edge to support uropod retraction and internalization (Alon and Dustin, 2007; Arnaout et al., 2005; Broussard et al., 2008; Calderwood, 2004; Evans and Calderwood, 2007; Luo et al., 2007).
Integrin αIIbβ3, the most abundant receptor on platelets, binds to fibrinogen and von Willebrand factor, and mediates platelet aggregation and association with injured vessel walls. Inherited mutations in its αIIb or β3 subunits result in the bleeding disorder Glanzmann's thrombasthenia. RGD-mimetic small molecules and an antibody to αIIbβ3 are prescribed for the prevention of thrombosis ((Springer et al., 2008; Xiao et al., 2004).
The integrin α and β subunits have large N-terminal extracellular domains, single-pass transmembrane domains, and usually short C-terminal cytoplasmic domains. The first crystal structure of an integrin ectodomain, of αVβ3, represented a huge advance (Xiong et al., 2001; Xiong et al., 2002). Together with subsequent work, ten of twelve domains in the ectofragment were revealed in a bent conformation (Xiao et al., 2004; Xiong et al., 2004). A ligand-binding head formed by both subunits is followed by legs in each subunit that connect to the transmembrane domains. There is an extreme bend at knees between the upper and lower legs. Integrin epidermal growth factor-like (I-EGF) domains 1 and 2 at the β-knee were disordered in the previous αVβ3 structure. Crystals of β2 leg fragments containing I-EGF domains 1 and 2 have been solved in two different orientations (Shi et al., 2007), but the conformation of these domains in the bent integrin conformation remains unknown.
Subsequent to the αVβ3 crystal structure, mutational studies on cell surface integrins and EM studies on αVβ3, αLβ2, and αXβ2 integrins demonstrated that the bent conformation is the physiologically relevant, low affinity integrin conformation (Nishida et al., 2006; Takagi et al., 2002). Nonetheless, a cryo EM study on αIIbβ3 revealed a different, less compact conformation with a different arrangement of leg domains (Adair and Yeager, 2002). Furthermore, two recent studies have revealed extended conformations of αIIbβ3 but failed to find a bent conformation (Rocco et al., 2008; Ye et al., 2008). Crystal structure studies on αIIbβ3 are important to resolve these controversies. Revealing the structure within a complete ectodomain of the bent β-knee is important to understanding the mechanism of integrin extension. Moreover, no integrin crystal structure to date has described the bent structure in the light of current knowledge that it is physiologically relevant, is in a low-affinity state, and with the aim of understanding how the bent conformation is stabilized and how it transitions to extended conformations. The previous αVβ3 bent conformation was described as “not expected to occur in the membrane-bound receptor,” and being in “its active (ligand competent) state” (Xiong et al., 2001).
Most studies find that upon activation, integrins extend. Upon extension, the headpiece can remain in the closed conformation, as when bent, or transition to an open conformation with high affinity for ligand (Xiao et al., 2004). In contrast, a “deadbolt model” posits that activation can occur in the absence of extension (Arnaout et al., 2005). Binding of cytoskeletal proteins such as talin and kindlins to the integrin β cytoplasmic domain appears to interfere with α/β cytoplasmic domain association, and induce integrin extension (Wegener and Campbell, 2008). However, there is currently no known feature of integrin structure that would enable cytoskeleton binding to couple to the extended, open conformation with high affinity for ligand. This would appear to be important to fulfill the key role of integrins in integrating the extracellular and intracellular environments.
Three closely linked metal ion binding sites in the β I domain are especially important in ligand binding. Mg2+ at the central, metal ion-dependent adhesion site (MIDAS) site directly coordinates the acidic sidechain shared by all integrin ligands. However, in previous unliganded, bent αVβ3 structures, the MIDAS and one adjacent site were unoccupied, and it was proposed that metal binding was either caused by integrin activation or induced by ligand binding (Xiong et al., 2002) However, crystals have not been reported with a combination of the two metal ions important for integrin ligand binding, Mg2+ and Ca2+. Therefore, in current comparisons between low and high affinity β I domain conformations, the changes associated with ligand binding and metal binding cannot be deconvoluted.